Multi-resistant Enterococcus faecium represent one of the most dangerous challenges in infectious diseases therapeutics. In particular, the widespread resistance to ??lactam antibiotics represents a compromise of our most effective anti-enterococcal agents. Although E. faecium resistance to ??lactams is attributed to expression of low-affinity class B penicillin binding protein Pbp5, our work has revealed that E. faecium cephalosporin susceptibility can be achieved through the deletion of two class A Pbps (PonA and PbpF), despite expression of Pbp5 and continued resistance to other ?-lactams. Moreover, the cephalosporin susceptibility of these deletion mutants can be reversed in association with expression of a novel protein (P5AP), whose expression appears to be related to a eukaryotic-like serine-threonine kinase-phosphatase system (stk-stpA) and whose deletion from a wild-type strain results in a cephalosporin- susceptible phenotype similar to that observed with deletion of ponA and pbpF. We have also shown that ??lactam resistance can be achieved in an E. faecium strain lacking Pbp5 through the activity of Ldtfm, an L,D-transpeptidase insensitive to inhibition by penicillins and cephalosporins. The expression of this resistance is also tied to stk-stpA. In the present proposal, we aim to characterize the structure and function of P5AP, examine its interaction with ceftriaxone and with Pbp5 and the class A Pbps and determine the location of its interaction with Pbp5. We will also examine the role of stk-stpA in the expression of cephalosporin resistance in strains lacking ponA and pbpF and in ampicillin resistance expressed by E. faecium lacking pbp5. Finally, we will investigate the interactions between Ldtfm and its acyl donor and compare these with previously characterized acylation reactions with carbapenems. Through this work we will expand our knowledge of ??lactam resistance mechanisms and cell wall physiology in E. faecium, characterize structure and function of novel proteins and lay the groundwork for development of novel therapeutic strategies against multi-resistant E. faecium.